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Sample GSM7659355

Query DataSets for GSM7659355

Status

Public on Aug 30, 2023

Title

Cell line Exon13.23, DMSO, Replicate 1

Sample type

SRA

Source name

PL2F7/F7

Organism

Mus musculus

Characteristics

cell line: PL2F7/F7
cell type: Embryonic stem cell
treatment: DMSO-Day14

Extracted molecule

genomic DNA

Extraction protocol

Genomic DNA was extracted from the cell pellets using Zymo Genomic DNA Extraction kit
DNA libraries were prepared using TruSeq Nano DNA Library Prep kit

Library strategy

OTHER

Library source

genomic

Library selection

other

Instrument model

Illumina MiSeq

Description

Sahu et al_Suppl file 2.xlsx

Data processing

The reads were demultiplexed using bcl2fastq (Illumina) and fastq files were generated. The paired end reads were merged using FLASH and reads were aligned to the reference amplicon with a global sequence alignment algorithm using Needleman-Wunsch Alignment.
The total number of aligned reads were quantified using CRISPResso2 http://crispresso.pinellolab.org/submission (63) and individual unique alignments were annotated with a custom-made variant caller modified from ANNOVAR (64) (release 2019-10-21) and the R/Bioconductor Biostrings package in R version 4.1.2 https://bioconductor.org/packages/Biostrings. Merged reads containing “N” bases were removed from the analysis.
Abundances of SNVs were quantified when the reads contained the synonymous PAM modification (HDR marker) and no other substitution or indels were present. The variants generated at a low frequency (less than 1 in 105 reads in any one of the replicates) were excluded from further analysis to prevent erroneous variant categorization. A pseudo count of 1 was added to all reads and for every sample and at all the conditions. Read counts for each SNVs were then normalized to the total read coverage of the sequencing library.
Dropout or enrichment scores were calculated by taking the ratio of frequency of the variants at day 14 in DMSO or cisplatin or olaparib over day 3. The scores were expressed in Log2 scale, which we define as function scores of SNVs in DMSO, cisplatin and olaparib.
The FSs, averaged over two independent replicates, were fit using a probit regression model for statistical modelling to calculate the probabilities of impact on function
Assembly: GRCh38
Supplementary files format and content: xlsx: Excel files include PIF, function scores, and additional annotations for all variants.
Library strategy: Amplicon sequencing

Submission date

Jul 24, 2023

Last update date

Aug 30, 2023

Contact name

Shyam Sharan

E-mail(s)

sharans@mail.nih.gov

Organization name

Mouse Cancer Genetics Program

Street address

Building 560, Room 32-33